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Related Experiment Videos

Bacterial selenocysteine synthase--structural and functional properties

P Tormay1, R Wilting, F Lottspeich

  • 1Lehrstuhl für Mikrobiologie, Universität München, Germany.

European Journal of Biochemistry
|August 4, 1998
PubMed
Summary

Selenocysteine synthase, a pyridoxal-5'-phosphate-dependent enzyme, uses Lys295 for activity. This enzyme specifically incorporates selenide over sulfur, crucial for selenocysteine incorporation into proteins.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • Selenocysteine synthase (SecS) from Escherichia coli is a pyridoxal-5 omino-phosphate (PLP)-dependent enzyme.
  • SecS catalyzes the conversion of seryl-tRNA(Sec) to selenocysteyl-tRNA(Sec).
  • SecS belongs to the alpha/gamma superfamily of PLP-dependent enzymes.

Purpose of the Study:

  • To identify the specific lysine residue in selenocysteine synthase responsible for binding the pyridoxal-5 omino-phosphate prosthetic group.
  • To investigate the substrate specificity of selenocysteine synthase, particularly its discrimination between selenium and sulfur compounds.

Main Methods:

  • Gene cloning and sequencing of selenocysteine synthases from Moorella thermoacetica and Desulfomicrobium baculatum.
  • Site-directed mutagenesis of conserved lysine residues to asparagine.

Related Experiment Videos

  • Enzyme activity assays and kinetic analysis.
  • Proteolytic fragmentation, mass spectrometry, and peptide sequencing.

    • Main Results:

    • A conserved lysine residue, Lys295, was identified as essential for selenocysteine synthase activity.
    • Mass spectrometry confirmed that Lys295 is modified by the pyridoxal-5 omino-phosphate prosthetic group.
    • The enzyme exhibited significantly lower catalytic efficiency with thiophosphate (forming cysteyl-tRNA(Sec)) compared to selenophosphate.
    • Selenide and sulfide could be used as substrates, but selenophosphate specificity was observed.

    Conclusions:

    • Lys295 is the critical residue for pyridoxal-5 omino-phosphate binding and catalysis in selenocysteine synthase.
    • The phosphate moiety of selenophosphate is key to the enzyme's specificity for selenium over sulfur.
    • These findings elucidate the mechanism of selenocysteine biosynthesis and enzyme specificity.